The human immunoglobulins (Igs), a class of plasma proteins produced by the immune system as a response to parasitic invasion, can be divided into five classes, IgG, IgA, IgM, IgD, and IgE. Human IgG (HIgG) has an average concentration of 12 mg/ml in adult blood, and encompasses 75% of all human immunoglobulins. HIgG consists of four subclasses IgG1, 2, 3, and 4. Among these subclasses, IgG1 is the most dominant protein, making up anywhere between 43 to 75% of all the HIgG (J. Harris, Blood Separation and Plasma Fractionation, John Wiley & Sons, New York, 325p (1990)). The HIgG backbone consists of two identical “heavy” chains and two identical “light” chains (FIG. 1). Disulfide bonds form bridges between chains, which confer flexibility to the HIgG molecule.
HIgG is usually obtained from pooled plasma by the Cohn-Oncley process, which needs at least 4 steps to achieve an IgG purity of 85%. HIgG isolated by this technique tends to aggregate to form multimers (J. Harris, Blood Separation and Plasma Fractionation, John Wiley & Sons, New York, 325p (1990)). Monoclonal antibodies produced in cell culture from hybridoma cells is another source for the production of HIgG. Diafiltered supernatant of cell culture can be subjected to different processes, such as ammonium sulfate precipitation, ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), or affinity chromatography (J. Harris, Blood separation and plasma fractionation, New York: A John Wiley & Sons 325 p. (1990)) to purify HIgG. Ion exchange chromatography (R. Necina et al., Biotechnology and Bioengineering 60(6):689-698 (1998)) has been used to purify HIgG directly from cell culture supernatant containing 3% fetal calf serum (FCS) yielding a recovery of less than 70% and a HIgG:BSA ratio of 1:2. Ammonium sulfate precipitation generated even less purity than ion-exchange chromatography (J. Harris, Blood separation and plasma fractionation, New York: A John Wiley & Sons. 325 p. (1990)). Pretreatment of the cell culture supernatant is necessary prior to using HIC, due to the high salt concentration usually found in the supernatant. Hydrophobic charge induction chromatography (HCIC) (L. Guerrier, et al., Journal of Chromatography B 755(1-2):37-46 (2001); L. Guerrier, et al., Bioseparation 9(4):211-221 (2000)) can be another choice of HIgG purification, yielding a purity of 98% when the feedstock solution was from protein-free cell culture supernatant. However, when dealing with cell culture supernatant containing 5% FCS, an IEC step had to be coupled to HCIC, and the purity and recovery of HIgG were found to be 69% and 76% respectively.
Among the chromatographic methods, affinity chromatography deserves particular attention because, in principle, it allows the possibility of obtaining several fold purification with high recovery in a single step. Protein A and protein G are the most commonly used ligands in the purification of HIgG. However, several drawbacks, including their high cost, low stability, and the possibility of contaminating products, make it important to find a new ligand for IgG purification. Synthetic small ligands may overcome some of these disadvantages and have been studied by many research groups. Table 1, below, lists small ligands that have been used in HIgG affinity purification.
Table 1. Small ligands used in the purification of IgG.
TABLE 1Small ligands used in the purification of IgG.IgGPurity/CompanyLigandSupportCapacitySourceElutionRecovery1.Chinese Academy ofsulfamethazinePoly(glycidyl methacrylate)HBP0.05M PBS, 0.25 NaCl,90%/Sciences, ChinapH 5.52.HoffmannLaRocheEPIHRSTLTALLAmino-NuGel320 μg/gCulture0.2M NaCl/0.2M HOAcInc. USA(SEQ ID NO:35)gel3.Research Institute forMannosylerythritol lipidPoly(2-hydroxyethyl106 mg/g0.1M Acetate buffer,/80%Green Technology,methacrylate)gelpH 3.6Japan4.Tecnogen SCpA,MAPEpoxy-activated EupergifAnti-0.1M acetic acidBiopharmaceuticals,AV13: GFRKYLHFRRHLLC30NMAPItaly(SEQ ID NO:33)sera(AV13)4K2KG(SEQ ID NO:36)AV15:VRLGWLLAPADLDAR(SEQ ID NO:37)(AV15)4(KRG)2KG(SEQ ID NO:38)5.Tecnogen, ItalyTG19318 (PAM):Epoxy-activated Eupergif25 mg/ml0.1M acetic acid(RTY)4K2KGC30Ngel(SEQ ID NO:39)6.Tecnogen, ItalyD-PAM, TG19320:Poly-acrylamide/azlactone-Serum0.1M acetic acid, pH 3.595%/KAPTIV-GYactivated gel (Emphaze(SEQ ID NO:40)matrix)7.AmershamProtein A4% Cross-link agarose20 mg/ml0.1M citrate acid, pH 3.0Biosciencesmoist gel8.AmershamProtein GSepharose high0.1M Gly, pH 2.7Biosciencesperformance9.Prometic BiosciencesMAbsorbent A1P/A2P6% Cross-link agarose50 mg/ml10/50 mM citrate buffer,Ltd.resinpH 3.010.University of22/8: Aromatic aminesSepharose 6B51.9 g/gHBP0.1M glycine-HCl, pH97.3/67%Cambridge, UKmoist gel2.9(0.025M citric acid, pH2.4)11.Hacettepe University,Transition Metal ionsPoly(hydroxyethyl50 mMTurkeymethacrylate)ethylenediaminetetraaceticacid (EDTA), pH 8.0